Cfx96 touch real time pcr detection system cfx96 manager version 3

Seedlings were synchronized under LD cycles in MS3 medium plates for 12–14 days and subsequently transferred to LL. Shoots and roots from intact plants were taken every 4 hour over the circadian cycle. For excised roots, shoots and roots were carefully separated with a sterile razor blade and the excised roots were deposited on MS3 agar medium plates for 2 or 3 days as specified. RNA was purified using a Maxwell RSC Plant RNA kit following the manufacturer’s recommendations (Promega). Single-stranded cDNA was synthesized using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad). qPCR analyses were performed with cDNAs diluted 50‐fold with nuclease‐free water using Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent) with a 96-well CFX96 Touch Real-Time PCR detection system (Bio-RAD CFX96 Manager version 3.1, Bio-Rad). Each sample was run in technical triplicates. The expression of PP2AA3 (PROTEIN PHOSPHATASE 2A SUBUNIT A3, AT1G13320) or MON1 (MONENSIN SENSITIVITY1, AT2G28390)^{67 (link)} was used as a control. Crossing point (Cp) calculation was used for quantification using the Absolute Quantification analysis by the 2^nd Derivative Maximun method. At least two biological replicates were performed, with measurements taken from distinct samples grown and processed at different times.